Question

Compare and contrast B-galatosidase, luciferase, and green fluorescent protein utilized as reporter systems. (pros and cons and general mechanism included)

Answer 1

Reporter system is important to describing the Eukaryotic gene expression and gene regulation study. This system indicators of transcriptional activity in cells for gene expression.

Under gene reporter system, reporter gene presents together with the promoter sequence in the expression vector. This expression vector transferred in the cell and when it expresses in the host, we can measure the amount of reporter mRNA, proteins and enzymatic activity of the reporter gene also. The ideal reporter system has characteristics like reproducible, rapid, sensitive and safe.

Presently many reporter genes and assays have been developed most of them are β -galactosidase, luciferase, growth hormone (GH), β-glucuronidase (GUS), alkaline phosphatase (AP), and green fluorescent protein (GFP).

1. β -galactosidase: The lacZ gene which codes for the β -galactosidase enzyme from E. coli. It is a Tetrameric enzyme with a subunit size of 1,16,000 Dalton.

• Commonly usable assay
• Easily expressible
• After expression, cell extract used
• Broadly used for in situ staining

Limitation:

• Endogenous activity
• Lower sensitivity in non-chemiluminescent assay

2. Luciferase: Luciferase enzyme used frequently for reporter gene technology. Its coding sequence is the luc gene cloned from the Photinus pyralis (firefly). It is a monomeric unit of 60,700 Dalton.

• Fast
• Easily expressible
• High sensitivity
• Large linear rang

Limitation:

• Requires lummo-meter for high sensitivity.

3. Green fluorescent protein (GFP): The green fluorescent protein isolated from the Aequorea Victoria (jellyfish). It consists of 238 ammo acids, 27000 Dalton monomer that emits green light when excited with UV or blue light.

• No substrates required
• Stable reporter protein in-situ and in-vivo application

Limitation:

• Low sensitivity
• Expression Expensive
• Microscope required